Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress, and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of exploration: (a) Antioxidant role of the cyclic oxidation and reduction of protein methionine residues. Reactive oxygen-mediated oxidation of methionine residues of proteins leads to the formation of a racemic mixture of the R- and S- stereo isomers of methionine sulfoxide (MetO). In previous studies, we showed that bacteria, yeast, and mammals contain an enzyme, methionine sulfoxide reductase (MsrA), that can catalyze reduction of the S-isomer of MetO back to methionine, and that mutant strains of mice lacking MsrA are more sensitive to oxidative stress-induced protein image and have a shorter maximum life span than the wild type strain. In continuing studies, we identified a second form of methionine sulfoxide reductase (MsrB) that is specific for the reduction of the R-isomer of MetO. We found further that in contrast to MsrA this R-specific reductase contains a selenocysteine residue at the catalytic site. By means of site-directed mutagenesis we expressed the mouse seleno-MsrB in Escherichia coli, demonstrated that substitution of cysteine for selenocysteine in this enzyme leads to a substantial (>90%) decrease in catalytic activity, and that replacement of the selenocysteine with either alanine or serine leads to complete loss of activity. In further studies, we will attempt to determine the effects of simultaneous deletions and overexpressions of both forms of reductase in mice on their longevity and sensitivity to oxidative stress. (b) Regulation of methionine sulfoxide transcription and /or translation. To identify proteins that are involved in the regulation of MsrA protein expression, partially purified nuclear proteins from both wild type and null mutant strains of yeast were tested for their ability to bind to the promoter region of the methionine sulfoxide reductase gene (msrA). The elongation factor (EF)-gamma, nuclear G-protein, and elongation factor-3, were among those that exhibited greater binding activity to msrA promoter DNA from the null mutant as compared to that from the wild-type. The elongation factor-1gamma was shown to bind to the 39 base pair segment at the 3'-prime end of the msrA promoter. Its role in the regulation of MsrA synthesis was confirmed by the demonstration that after their transfection with the msrA gene together with its promoter region, the level of MsrA in a wild type yeast strain was three-fold greater than that in a mutant strain lacking factor 1-gamma. (c) Role of Caspase-12 in cell signaling and manganese-induced apoptosis. We reported earlier that high concentrations of manganese induces apoptosis in 3T3 cells by a non-mitochondrial-mediated mechanism in which caspase 12 plays a key role. To elucidate basic mechanisms involved in the regulation of caspase-12 gene regulation, we have now isolated the DNA fragments containing the basal promoter and the regulatory element responsive to serum starvation in rat caspase-12 expression. In addition, the transcriptional start site of caspase-12 has been identified. Further studies are directed toward identification of the growth factors, cytokines, or transcription factors in serum that regulate expression of this enzyme. Furthermore, the caspase 12 gene has been over expressed in the Pichi pasterns yeast where it is excreted into the growth medium, from which it is being purified. We hope to be able to crystallize the protein and examine its ability to interact with potential regulatory proteins known to be implicated in apoptosis. (d) Inflammation-induced oxidation of methionine residues of proteins. The biosynthesis of hypochlorous acid by neutrophils and macrophage represents a major mechanism for antibacterial action in mammals. Because methionine residues of proteins are particularly sensitive to oxidation by hypochlorous acid, we have carried out studies aimed at elucidation of the mechanism involved. As noted in last years report, preliminary results demonstrated that chloramine derivatives are intermediates. However, further studies have shown that other mechanisms not yet elucidated are also involved. (e) Metal-catalyzed oxidation of proteins in aging and disease. Previous studies in this laboratory led to the demonstration that metal-catalyzed oxidation of proteins is the major mechanism involved in the oxidation of proteins to carbonyl derivatives, and that lysine and arginine residues are major targets for these metal-catalyzed reactions. We have now initiated a program to generate antibodies against oxidized forms of arginine and lysine residues that can be used to examine the contribution of metal-catalyzed oxidation to aging and various diseases.